In the practice of biotechnology, nucleic acid fragments are commonly isolated from prokaryotic and eukaryotic and viral cultures. The isolation of these fragments enables their sequencing, their use as probes for diagnostic and other research assays, and their assembly into genes encoding whole proteins or polypeptides. Traditionally, nucleic acids have been separated from contaminating proteinaceous material (e.g., from bacterial and viral cultures) by lysis in the presence of a detergent (e.g., sodium dodecyl sulfate) and a salt solution (e.g., potassium acetate) followed by extraction (deproteinization) with phenol or chloroform or a mixture thereof. These procedures separate the nucleic acids, generally by precipitation, from lipid and protein contaminants of the cell or virus culture.
In recombinant DNA research, DNA molecules are commonly isolated from bacterial cell cultures and bacteriophage cultures. For example, double-stranded plasmid DNA is produced within and isolated from bacterial cells, e.g. Escherichia coli, that are cultured in liquid nutrient broth media. Bacteriophage M13 single-stranded template DNA is produced by the bacteriophage M13 propagated on an appropriate E. coli host. Template DNA is isolated from bacteriophage that have been released by host bacteria into a nutrient broth medium. The isolation of these plasmid DNA or template DNA molecules enables the sequencing thereof, and the use of the molecules for diagnostic and other assays, for their assembly into genes encoding a polypeptide of interest or for their use as vectors to produce such polypeptides.
The procedure commonly used for the isolation of plasmid DNA from bacterial cultures is described in H. C. Birnboim and J. Doly, "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucleic Acids Res., 7: 1513-1523 (1979). Template DNA is isolated in a similar manner. See, e.g., Leder et al., Science, 196:175 (1977). Briefly, the DNA is separated from contaminating proteinaceous material in the conditioned medium by lysis in the presence of lysozyme in a detergent and salt solution. This lysis step is followed after an incubation or neutralization step at a low temperature by extraction or deproteinization with phenol or chloroform or a mixture thereof. The nucleic acids are then separated by precipitation from lipid and other protein constituents of the culture. In non-plasmid, cosmid or phagmid DNA separation techniques, the neutralization step is typically not needed. In the procedures, lysis and deproteinization, or neutralization and deproteinization, are separately performed in two distinct steps because the reagents needed to perform the lysis or neutralization steps are not miscible with the reagents involved in deproteinization. See also: T. Maniatis et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory (1982) for a more detailed description of the specific steps of the isolation procedure.
Due to the frequency with which these DNA isolating and purifying steps are employed, and the researcher time which is consumed in performing the Maniatis et al. steps, there is a need in the art of recombinant DNA research for more efficient methods of DNA isolation.